3_Maintenance and Preservation of Microbial Cultures

20-30 days

Store cultures in a refrigerator (4⁰C). Microbes remain viable for __ and must be subcultured again on a fresh media.

• risks of contamination,

• transposition of strain numbers or designations due to mislabeling, possible loss of culture,

• lack of required storage space

• the higher possibility of genetic changes through spontaneous mutation resulting into phenotypic variations of the original culture

This maintenance method may have some disadvantages such as:

8-10 ml

After incubation, add about ___sterile mineral oil or liquid paraffin to cover the growth on the agar slant and up to 1 cm above the tip of the slant.

1 to 2 years

Some microbes have been preserved satisfactorily for a period of up to ___by Overlaying cultures with mineral oil

Conservation

is the act of preserving, guarding, or protecting.

lost or damaged

The objective is to protect microorganisms from being___from something which can harm it

(in situ conservation and ex situ preservation

There are two types of strategies that help to improve the incorporation of microbial communities and ecosystems into conservation agenda.

ex situ preservation

• Isolation, handling and preservation of microorganisms in the culture collections is known as ___  which is widely being followed throughout world.

• incorporates the microbial taxa and communities into preservation programme. 

• Proper maintenance and preservation of microorganism’s ex situ requires a superior laboratory facility with all the requisite equipments.

• includes the gene banks, culture collections, and microbial resources centers making the repository for microbial isolates and keeps away from the time-consuming re isolation protocols.

• to retain the viability of the stock culture for a long period of time while maintaining its purity and trait of being “true-to-type”

Isolation, handling and preservation

___of microorganisms in the culture collections is known as ex situ preservation which is widely being followed throughout world.

In situ conservation

includes on site conservation of the microbial flora along with their ecosystem and habitat and the maintenance as well as recovery.

Short-term preservation, Medium-term preservation, Long-term preservation

Methods of Preservation (duration)

Short-term preservation

• refrigeration

• Periodic transfer to fresh media – grown in slants, incubated and then refrigerated at 4C

• Active culture sealed with parafilm (tubes and plates)

• Considerations

  • Time interval of transfers

  • proper medium

  • proper storage temperature

Medium-term preservation

• Mineral oil,

• Silica gel (fungal spores ),

• Soil (fungal),

• Sterile water

• 10-12 years

• Prevents evaporation from cultures and limit availability of O2 to decrease metabolic rate

• Advantages

  • Simple

  • enables one to remove some growth under the oil and inoculate it in a fresh medium and still preserve the initial culture 

• Disadvantage

  • viability of microorganisms varies with species (some fungi still grows)

Long-term preservation

• Ultralow Freezing (Ultralow freezers)

• Cryogenic Freezing (Cryogenic Freezers)

• Freeze-Drying (Lyophilization)

• Employs

  • rapid drying in frozen state

  • dry ice in alcohol

• Advantages

  • long-term survival

  • less opportunity for changes in the characteristics of culture

  • smallness of storage containers

Freeze-drying (lyophilization)

• long-term preservation

• temperature = -70oC

• Water is removed by sublimation (Primary drying by vacuum) then desorption (secondary drying)

• includes lyoprotectants (glycerol)

Ultra low freezing

• long-term preservation

• freezing with temperature -80oC

• Considerations

  • cryoprotective agent (glycerol)

Cryogenic freezing

• long-term preservation

• freezing with liquid nitrogen

  • temperature = -196oC

  • Cells trapped in glass like matrix

  • Same effect as ultra freezing

• Considerations

• Cryoprotectants penetrating both cell wall and cytoplasmic membrane, i.e. DMSO and glycerol.

• cryotank

Drying

Culture Preservation Methods:

• drying temperature = 45oC

• Limitation:

  • for spore- and cyst- formers

Culture Collections

• organizations which maintain authentic pure cultures of microorganisms

• provide ‘type’ strains to microbiologists throughout the world

• American Type Culture Collection (ATCC) (Maryland)

• National Collection of Type Cultures (NCTC) (London)

• Japanese Type Culture Collection (JTCC) (Japan)

• Philippine National Collection of Microorganisms (PNCM) (BIOTECH-UPLB

Examples of BANKING MICROBES